The present invention relates generally to the aging of proteins resulting from their reaction with glucose and other reducing sugars, and more particularly to the inhibition of the reaction of nonenzymatically glycosylated proteins and the often resultant formation of advanced glycosylation (glycation) endproducts and cross-links.
The reaction between glucose and proteins has been known for some time. Its earliest manifestation was in the appearance of brown pigments during the cooking of food, which was identified by Maillard in 1912, who observed that glucose or other reducing sugars react with amino acids to form adducts that undergo a series of dehydrations and rearrangements to form stable brown pigments. Further studies have suggested that stored and heat treated foods undergo nonenzymatic browning as a result of the reaction between glucose and the polypeptide chain, and that the proteins are resultingly cross-linked and correspondingly exhibit decreased bioavailability.
This reaction between reducing sugars and food proteins was found to have its parallel in vivo. Thus, the nonenzymatic reaction between glucose and the free amino groups on proteins to form a stable, 1-deoxyketosyl adduct, known as the Amadori product, has been shown to occur with hemoglobin, wherein a rearrangement of the amino terminal of the beta-chain of hemoglobin by reaction with glucose, forms the adduct known as hemoglobin Alc. The reaction has also been found to occur with a variety of other body proteins, such as lens crystallins, collagen and nerve proteins. See Bucala et al., xe2x80x9cAdvanced Glycosylation; Chemistry, Biology, and Implications for Diabetes and Agingxe2x80x9d in Advances in Pharmacology, Vol. 23, pp. 1-34, Academic Press (1992).
Moreover, brown pigments with spectral and fluorescent properties similar to those of late-stage Maillard products have also been observed in vivo in association with several long-lived proteins, such as lens proteins and collagen from aged individuals. An age-related linear increase in pigment was observed in human dura collagen between the ages of 20 to 90 years. Interestingly, the aging of collagen can be mimicked in vitro by the cross-linking induced by glucose; and the capture of other proteins and the formation of adducts by collagen, also noted, is theorized to occur by a cross-linking reaction, and is believed to account for the observed accumulation of albumin and antibodies in kidney basement membrane.
In U.S. Pat. No. 4,758,583, a method and associated agents were disclosed that served to inhibit the formation of advanced glycosylation endproducts by reacting with an early glycosylation product that results from the original reaction between the target protein and glucose. Accordingly, inhibition was postulated to take place as the reaction between the inhibitor and the early glycosylation product appeared to interrupt the subsequent reaction of the glycosylated protein with additional protein material to form the cross-linked late-stage product. One of the agents identified as an inhibitor was aminoguanidine, and the results of further testing have borne out its efficacy in this regard.
While the success that has been achieved with aminoguanidine and similar compounds is promising, a need continues to exist to identify and develop additional inhibitors that broaden the availability and perhaps the scope of this potential activity and its diagnostic and therapeutic utility.
In accordance with the present invention, a method and compositions are disclosed for the inhibition of the advanced glycosylation of proteins (protein aging). In particular, the compositions comprise agents for inhibiting nonenzymatic cross-linking (protein aging) due to the formation of advanced glycosylation (glycation) endproducts. The agents may be selected from those materials capable of reacting with an early glycosylation product from the reaction of glucose with proteins and preventing further reactions. Cross-linking caused by other reactive sugars present in vivo or in foodstuffs, including ribose, galactose and fructose would also be prevented by the methods and compositions of the present invention.
The agents comprise substituted triazine compounds having the following structural formula: 
wherein R is hydrogen or an amino group;
Rxe2x80x2 is a cycloalkyl group;
an aralkyl group wherein the alkyl portion contains from 1 to 6 carbon atoms, and the aryl portion is optionally substituted by one to three hydroxy, lower alkoxy or nitro groups;
a heteroaryl group containing from 1 to 3 heteroatoms selected from the group consisting of sulfur, oxygen and nitrogen;
a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group; or
a lower alkyl or alkylene group, optionally substituted by hydroxy or a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group;
and their biologically or pharmaceutically acceptable acid addition salts; and mixtures thereof, and a carrier therefor.
The compounds, and their compositions, utilized in this invention appear to react with an early glycosylation product thereby preventing the same from later forming the advanced glycosylation end products which lead to protein cross-links, and thereby, to protein aging.
The present invention also relates to a method for inhibiting protein aging by contacting the initially glycosylated protein at the stage of the early glycosylation product with a quantity of one or more of the agents of the present invention, or a composition containing the same. In the instance where the present method has industrial application, one or more of the agents may be applied to the proteins in question, either by introduction into a mixture of the same in the instance of a protein extract, or by application or introduction into foodstuffs containing the protein or proteins, all to prevent premature aging and spoilage of the particular foodstuffs.
The ability to inhibit the formation of advanced glycosylation endproducts carries with it significant implications in all applications where protein aging is a serious detriment. Thus, in the area of food technology, the retardation of food spoilage would confer an obvious economic and social benefit by making certain foods of marginal stability less perishable and therefore more available for consumers. Spoilage would be reduced as would the expense of inspection, removal, and replacement, and the extended availability of the foods could aid in stabilizing their price in the marketplace. Similarly, in other industrial applications where the perishability of proteins is a problem, the admixture of the agents of the present invention in compositions containing such proteins would facilitate the extended useful life of the same. Presently used food preservatives and discoloration preventatives such as sulfur dioxide, known to cause toxicity including allergy and asthma in animals, can be replaced with compounds such as those described herein.
The present method has particular therapeutic application as the Maillard process acutely affects several of the significant protein masses in the body, among them collagen, elastin, lens proteins, and the kidney glomerular basement membranes. These proteins deteriorate both with age (hence the application of the term xe2x80x9cprotein agingxe2x80x9d) and as a consequence of diabetes. Accordingly, the ability to either retard or substantially inhibit the formation of advanced glycosylation endproducts carries the promise of treatment for diabetes and, of course, improving the quality and, perhaps, duration of animal life.
The present agents are also useful in the area of personal appearance and hygiene, as they prevent the staining of teeth by cationic anti-microbial agents with anti-plaque properties, such as chlorhexidine.
Accordingly, it is a principal object of the present invention to provide a method for inhibiting the extensive cross-linking of proteins that occurs as an ultimate consequence of the reaction of the proteins with glucose and other reactive sugars, by correspondingly inhibiting the formation of advanced glycosylation endproducts.
It is a further object of the present invention to provide a method as aforesaid which is characterized by a reaction with an initially glycosylated protein identified as an early glycosylation product.
It is a further object of the present invention to provide a method as aforesaid which prevents the rearrangement and cross-linking of the said early glycosylation products to form the said advanced glycosylation endproducts.
It is a yet further object of the present invention to provide agents capable of participating in the reaction with the said early glycosylation products in the method as aforesaid.
It is a still further object of the present invention to provide therapeutic methods of treating the adverse consequences of protein aging by resort to the aforesaid method and agents.
It is a still further object of the present invention to provide a method of inhibiting the discoloration of teeth by resort to the aforesaid method and agents.
It is a still further object of the present invention to provide compositions including pharmaceutical compositions, all incorporating the agents of the present invention.
It is still further object of the present invention to provide novel compounds, as well as processes for their preparation, for use in the methods and compositions of the present invention.
Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing description.
In accordance with the present invention, agents, compositions including pharmaceutical compositions containing said agents and associated methods have been developed which are believed to inhibit the formation of advanced glycosylation endproducts in a number of target proteins existing in both animals and plant material.
In particular, the invention relates to a composition which may contain one or more agents comprising substituted triazine compounds having the structural formula: 
wherein R is hydrogen or an amino group;
Rxe2x80x2 is a cycloalkyl group;
an aralkyl group wherein the alkyl portion contains from 1 to 6 carbon atoms, and the aryl portion is optionally substituted by one to three hydroxy, lower alkoxy or nitro groups;
a heteroaryl group containing from 1 to 3 heteroatoms selected from the group consisting of sulfur, oxygen and nitrogen;
a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group; or
a lower alkyl or alkylene group, optionally substituted by hydroxy or a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group;
and their biologically or pharmaceutically acceptable acid addition salts; and mixtures thereof, and a carrier therefor.
The lower alkyl groups referred to above preferably contain 1-6 carbon atoms and include methyl, ethyl, propyl, butyl, pentyl, hexyl, and the corresponding branched-chain isomers thereof. The lower alkylene groups likewise contain from 2 to 6 carbon atoms, and are exemplified by ethylene, propylene, butylene, pentylene, hexylene, and the corresponding branched chain isomers thereof. These groups are optionally substituted by one or more hydroxy groups, or a group of the formula 
wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group.
These substituted carbonyl compounds are thus carboxy, alkoxycarbonyl, arylcarbonyl or hydrazinylcarbonyl compounds wherein the alkyl portion contains from 1 to 6 carbon atoms as described hereinabove. Similarly, the aryl portion is as defined hereinbelow.
The aryl groups encompassed by the above formula are those containing 6-10 carbon atoms, such as naphthyl, phenyl and lower alkyl substituted-phenyl, e.g., tolyl and xylyl, and are optionally substituted by 1-3 hydroxy, lower alkoxy or nitro groups. Preferred aryl groups are phenyl and methoxyphenyl groups.
Where the possibility exists for substitution of a phenyl or aryl ring, the position of the substituents may be ortho, meta, or para to the point of attachment of the phenyl or aryl ring to the nitrogen of the hydrazine group.
The cycloalkyl groups contain from 1 to 7 carbon atoms, and are typified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, optionally substituted by one or more lower alkyl groups.
The heteroaryl groups of the compounds of formula I typically contain from 5 to 6 ring atoms, of which 1 to 3 are selected from the group consisting of oxygen, nitrogen and sulfur. Representative of such groups are 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyrrolyl, and 3-pyrrolyl, optionally substituted by one or more lower alkyl groups.
The lower alkoxy groups contain 1-6, and preferably 1-3, carbon atoms and are illustrated by methoxy, ethoxy, n-propoxy, isopropoxy and the like.
For the purposes of this invention equivalent to the compounds of formula (I) are the biologically and pharmaceutically acceptable acid addition salts thereof. Such acid addition salts may be derived from a variety of organic and inorganic acids such as sulfuric, phosphoric, hydrochloric, hydrobromic, sulfamic, citric, lactic, maleic, succinic, tartaric, cinnamic, acetic, benzoic, gluconic, ascorbic, methanesulfonic and related acids.
Of the compounds encompassed by Formula I, certain substituents are preferred. For instance, the compounds wherein R is a lower alkyl group, and particularly those wherein R is a methyl group, are preferred.
Representative compounds of the present invention are:
3,4-diamino-6-methyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(4-hydroxybenzyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-ethyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-benzyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(2-thienyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(3,4-dimethoxystyryl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(2xe2x80x2-nitrobenzyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(2xe2x80x2-carboxyethyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-isopropyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(2-methypropyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-propyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-butyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(1-methylpropyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-cyclopropyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-phenethyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-carboxyethyl-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(2xe2x80x2-ethoxycarbonylethyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(1xe2x80x2-ethoxycarbonylethyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(1xe2x80x2-ethoxycarbonylheptyl)-1,2,4-triazine-5(4H)-one;
3,4-diamino-6-(3xe2x80x2-methoxyphenylcarbonylmethylene)-1,2,4-triazine-5(4H)-one;
6-(3,4-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide;
3-[6xe2x80x2-(3xe2x80x2,4xe2x80x2-diamino-5xe2x80x2(4H)-one-1xe2x80x2,2xe2x80x2,4xe2x80x2-triazinyl)]-propionic hydrazide;
2-methyl-6xe2x80x2-(3xe2x80x2,4xe2x80x2-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide;
3,4-diamino-6-(1xe2x80x2,1xe2x80x2-dimethyl-2xe2x80x2-hydroxyethyl)-1,2,4-triazine-5(4H)-one;
4-amino-3-hydrazino-6-methyl-1,2,4-triazine-5(4H)-one;
4-amino-3-hydrazino-6-(4-hydroxybenzyl)-1,2,4-triazine-5(4H)-one;
4-amino-3-hydrazino-1,2,4-triazine-5(4H)-one; and their biologically and pharmaceutically acceptable salts.
The above compounds are capable of inhibiting the formation of advanced glycosylation endproducts on target proteins. The cross-linking of the protein to form the advanced glycosylation endproduct contributes to the entrapment of other proteins and results in the development in vivo of conditions such as reduced elasticity and wrinkling of the skin, certain kidney diseases, atherosclerosis, osteoarthritis and the like. Similarly, plant material that undergoes nonenzymatic browning deteriorates and, in the case of foodstuffs, become spoiled or toughened and, consequently, inedible. Thus, the compounds employed in accordance with this invention inhibit this late-stage Maillard effect and intervene in the deleterious changes described above.
The rationale of the present invention is to use agents which block the post-glycosylation step, i.e., the formation of fluorescent chromophores, the presence of which chromophores is associated with, and leads to adverse sequelae of diabetes and aging. An ideal agent would prevent the formation of the chromophore and its associate cross-links of proteins to proteins and trapping of proteins on the other proteins, such as occurs in arteries and in the kidney.
The chemical nature of the early glycosylation products with which the compounds of the present invention are believed to react may vary, and accordingly the term xe2x80x9cearly glycosylation product(s)xe2x80x9d as used herein is intended to include any and all such variations within its scope. For example, early glycosylation products with carbonyl moieties that are involved in the formation of advanced glycosylation endproducts, and that may be blocked by reaction with the compounds of the present invention, have been postulated. In one embodiment, it is envisioned that the early glycosylation product may comprise the reactive carbonyl moieties of Amadori products or their further condensation, dehydration and/or rearrangement products, which may condense to form advanced glycosylation endproducts. In another scenario, reactive carbonyl compounds, containing one or more carbonyl moieties (such as glycolaldehyde, glyceraldehyde or 3-deoxyglucosone) may form from the cleavage of Amadori or other early glycosylation endproducts, and by subsequent reactions with an amine or Amadori product, may form carbonyl containing advanced glycosylation products such as alkylformyl-glycosylpyrroles.
The compositions useful in the present invention comprise or contain agents capable of reacting with the active carbonyl intermediate of an early glycosylation product. Suitable agents are the compounds of Formula I of the present invention. The present invention likewise relates to methods for inhibiting the formation of advanced glycosylation endproducts, which comprise contacting the target proteins with a composition of the present invention. In the instance where the target proteins are contained in foodstuffs, whether of plant or animal origin, these foodstuffs could have applied to them by various conventional means a composition containing the present agents.
In the food industry, sulfites were found years ago to inhibit the Maillard reaction and are commonly used in processed and stored foods. Recently, however, sulfites in food have been implicated in severe and even fatal reactions in asthmatics. As a consequence, the sulfite treatment of fresh fruits and vegetables has been banned. The mechanism for the allergic reaction is not known. Accordingly, the present compositions and agents offer a nontoxic alternative to sulfites in the treatment of foods in this manner.
As is apparent from a discussion of the environment of the present invention, the present methods and compositions hold the promise for arresting the aging of key proteins both in animals and plants, and concomitantly, conferring both economic and medical benefits as a result thereof. In the instance of foodstuffs, the administration of the present composition holds the promise for retarding food spoilage thereby making foodstuffs of increased shelf life and greater availability to consumers. Replacement of currently-used preservatives, such as sulfur dioxide known to cause allergies and asthma in humans, with non-toxic, biocompatible compounds is a further advantage of the present invention.
The therapeutic implications of the present invention relate to the arrest of the aging process which has, as indicated earlier, been identified in the aging of key proteins by advanced glycosylation and cross-linking. Thus, body proteins, and particularly structural body proteins, such as collagen, elastin, lens proteins, nerve proteins, kidney glomerular basement membranes and other extravascular matrix components would all benefit in their longevity and operation from the practice of the present invention. The present invention thus reduces the incidence of pathologies involving the entrapment of proteins by cross-linked target proteins, such as retinopathy, cataracts, diabetic kidney disease, glomerulosclerosis, peripheral vascular disease, arteriosclerosis obliterans, peripheral neuropathy, stroke, hypertension, atherosclerosis, osteoarthritis, periarticular rigidity, loss of elasticity and wrinkling of skin, stiffening of joints, glomerulonephritis, etc. Likewise, all of these conditions are in evidence in patients afflicted with diabetes mellitus. Thus, the present therapeutic method is relevant to treatment of the noted conditions in patients either of advanced age or those suffering from one of the mentioned pathologies.
Protein cross-linking through advanced glycosylation product formation can decrease solubility of structural proteins such as collagen in vessel walls and can also trap serum proteins, such as lipoproteins to the collagen. Also, this may result in increased permeability of the endothelium and consequently covalent trapping of extravasated plasma proteins in subendothelial matrix, and reduction in susceptibility of both plasma and matrix proteins to physiologic degradation by enzymes. For these reasons, the progressive occlusion of diabetic vessels induced by chronic hyperglycemia has been hypothesized to result from excessive formation of glucose-derived cross-links. Such diabetic microvascular changes and microvascular occlusion can be effectively prevented by chemical inhibition of advanced glycosylation product formation utilizing a composition and the methods of the present invention.
Studies indicate that the development of chronic diabetic damage in target organs is primarily linked to hyperglycemia so that tight metabolic control would delay or even prevent end-organ damage. See Nicholls et al., Lab. Invest., 60, No. 4, p. 486 (1989), which discusses the effects of islet isografting and aminoguanidine in murine diabetic nephropathy. These studies further evidence that aminoguanidine diminishes aortic wall protein cross-linking in diabetic rats and confirm earlier studies by Brownlee et al., Science, 232, pp. 1629-1632 (1986) to this additional target organ of complication of diabetes. Also, an additional study showed the reduction of immunoglobulin trapping in the kidney by aminoguanidine (Brownlee et al., Diabetes 35, Suppl. 1, p. 42A (1986)).
Further evidence in the streptozotocin-diabetic rat model that aminoguanidine administration intervenes in the development of diabetic nephropathy was presented by Brownlee et al., 1988, supra, with regard to morphologic changes in the kidney which are hallmarks of diabetic renal disease. These investigators reported that the increased glomerular basement membrane thickness, a major structural abnormality characteristic of diabetic renal disease, was prevented with aminoguanidine.
Taken together, these data strongly suggest that inhibition of the formation of advanced glycosylation endproducts (AGEs), by the teaching of the present invention, may prevent late, as well as early, structural lesions due to diabetes, as well as changes during aging caused by the formation of AGEs.
Diabetes-induced changes in the deformability of red blood cells, leading to more rigid cell membranes, is another manifestation of cross-linking and aminoguanidine has been shown to prevent it in vivo. In such studies, New Zealand White rabbits, with induced, long-term diabetes are used to study the effects of a test compound on red blood cell (RBC) deformability (df). The test compound is administered at a rate of 100 mg/kg by oral gavage to diabetic rabbits.
A further consequence of diabetes is the hyperglycemia-induced matrix bone differentiation resulting in decreased bone formation usually associated with chronic diabetes. In animal models, diabetes reduces matrix-induced bone differentiation by 70%.
In the instance where the compositions of the present invention are utilized for in vivo or therapeutic purposes, it may be noted that the compounds or agents used therein are biocompatible. Pharmaceutical compositions may be prepared with a therapeutically effective quantity of the agents or compounds of the present invention and may include a pharmaceutically acceptable carrier, selected from known materials utilized for this purpose. Such compositions may be prepared in a variety of forms, depending on the method of administration. Also, various pharmaceutically acceptable addition salts of the compounds of Formula I may be utilized.
A liquid form would be utilized in the instance where administration is by intravenous, intramuscular or intraperitoneal injection. When appropriate, solid dosage forms such as tablets, capsules, or liquid dosage formulations such as solutions and suspensions, etc., may be prepared for oral administration. For topical or dermal application to the skin or eye, a solution, a lotion or ointment may be formulated with the agent in a suitable vehicle such as water, ethanol, propylene glycol, perhaps including a carrier to aid in penetration into the skin or eye. For example, a topical preparation could include up to about 10% of the compound of Formula I. Other suitable forms for administration to other body tissues are also contemplated.
In the instance where the present method has therapeutic application, the animal host intended for treatment may have administered to it a quantity of one or more of the agents, in a suitable pharmaceutical form. Administration may be accomplished by known techniques, such as oral, topical and parenteral techniques such as intradermal, by subcutaneous, intravenous or intraperitoneal injection, as well as by other conventional means. Administration of the agents may take place over an extended period of time at a dosage level of, for example, up to about 30 mg/kg.
As noted earlier, the invention also extends to a method of inhibiting the discoloration of teeth resulting from nonenzymatic browning in the oral cavity which comprises administration to a subject in need of such therapy an amount effective to inhibit the formation of advanced glycosylation endproducts of a composition comprising an agent of structural Formula I.
The nonenzymatic browning reaction which occurs in the oral cavity results in the discoloration of teeth. Presently used anti-plaque agents accelerate this nonenzymatic browning reaction and further the staining of the teeth. Recently, a class of cationic anti-microbial agents with remarkable anti-plaque properties have been formulated in oral rinses for regular use to kill bacteria in the mouth. These agents, the cationic antiseptics, include such agents as alexidine, cetyl pyridinium chloride, chlorhexidine gluconate, hexetidine, and benzalkonium chloride.
Tooth staining by chlorhexidine and other anti-plaque agents apparently results from the enhancement of the Maillard reaction. Nordbo, J. Dent. Res., 58, p. 1429 (1979) reported that chlorhexidine and benzalkonium chloride catalyze browning reactions in vitro. Chlorhexidine added to mixtures containing a sugar derivative and a source of amino groups underwent increased color formation, attributed to the Maillard reaction. It is also known that use of chlorhexidine results in an increased dental pellicle. Nordbo proposed that chlorhexidine resulted in tooth staining in two ways: first, by increasing formation of pellicle which contains more amino groups, and secondly, by catalysis of the Maillard reaction leading to colored products.
In accordance with this method, the compounds of Formula I are formulated into compositions adapted for use in the oral cavity. Particularly suitable formulations are oral rinses and toothpastes incorporating the active agent.
In the practice of this invention, conventional formulating techniques are utilized with nontoxic, pharmaceutically acceptable carriers typically utilized in the amounts and combinations that are well-known for the formulation of such oral rinses and toothpastes.
The agent of Formula I is formulated in compositions in an amount effective to inhibit the formation of advanced glycosylation endproducts. This amount will, of course, vary with the particular agent being utilized and the particular dosage form, but typically is in the range of 0.01% to 1.0%, by weight, of the particular formulation.
Certain of the compounds encompassed by Formula I are novel compounds which can be prepared by modifications of chemical syntheses well-known in the art. These compounds are those compounds of formula I wherein the R group is an amino group, with the proviso that when Rxe2x80x2 is a lower alkyl group, it is substituted as defined in formula I hereinabove and the compounds of formula I wherein the R group is hydrogen, with the proviso that when Rxe2x80x2 is a lower alkyl group, it is substituted by hydroxy or a group of the formula 
wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group. These compounds can be represented by the structural formula (Ia): 
wherein when R is an amino group, then
Rxe2x80x2 is a cycloalkyl group;
an aralkyl group wherein the alkyl portion contains from 1 to 6 carbon atoms, and the aryl portion is optionally substituted by one to three hydroxy, lower alkoxy or nitro groups;
a heteroaryl group containing from 1 to 3 heteroatoms selected from the group consisting of sulfur, oxygen and nitrogen;
a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group; or
a lower alkyl or alkylene group, optionally substituted by hydroxy or a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group;
or when R is hydrogen; then
Rxe2x80x2 is a cycloalkyl group;
a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group; or a lower alkyl group or an alkylene-group, optionally substituted by hydroxy or a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, or a group of the formula
xe2x80x94NRxe2x80x2xe2x80x3NH2
wherein Rxe2x80x2xe2x80x3 is hydrogen or a methyl group;
with the proviso that when Rxe2x80x2 is a lower alkyl group, then it must be a substituted;
and their biologically or pharmaceutically acceptable acid addition salts.
The following compounds of the present invention are thus novel and heretofore unknown in the prior art:
3,4-diamino-6-(3,4-dimethoxystyryl)-1,2,4-triazine-5(4H)-one hydrochloride;
3,4-diamino-6-(2xe2x80x2-carboxyethyl)-1,2,4-triazine-5(4H)-one hydrochloride;
3,4-diamino-6-cyclopropyl-1,2,4-triazine-5(4H)-one hydrochloride;
3,4-diamino-6-carboxyethyl-1,2,4-triazine-5(4H)-one hydrochloride;
3,4-diamino-6-(2xe2x80x2-ethoxycarbonylethyl)-1,2,4-triazine-5(4H)-one hydrochloride;
3,4-diamino-6-(2xe2x80x2-ethoxycarbonylethyl)-1,2,4-triazine-5(4H)-one hydrochloride;
3,4-diamino-6-(1xe2x80x2-ethoxycarbonylheptyl)-1,2,4-triazine-5(4H)-one hydrochloride;
3,4-diamino-6-(3xe2x80x2-methoxyphenylcarbonylmethylene)-1,2,4-triazine-5(4H)-one;
6-(3,4-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide;
6-(3,4-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide hydrochloride;
3-[6xe2x80x2-(3xe2x80x2,4xe2x80x2-diamino-5xe2x80x2(4H)-one-1xe2x80x2,2xe2x80x2,4xe2x80x2-triazinyl)]-propionic hydrazide;
2-methyl-6xe2x80x2-(3xe2x80x2,4xe2x80x2-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide;
3,4-diamino-6-(1xe2x80x2,1xe2x80x2-dimethyl-2xe2x80x2-hydroxyethyl)-1,2,4-triazine-5(4H)-one hydrochloride; and
4-amino-3-hydrazino-6-(4-hydroxybenzyl)-1,2,4-triazine-5(4H)-one.
The compounds of formula I can be prepared according to the methods described in Dornow et al., Chem. Ber. 97:2647 and 2173 (1964), Veda et al., Chem. Pharm. Bul. (1964), 12, 100, J. Chem Soc., Perkin Trans., 1(12), 2037-2049 (1986). or those of U.S. Pat. Nos. 4,036,632 and 3,961,936, or as shown in the various schemes below. These references, in particular, describe the preparation of the compounds of formula I wherein R is hydrogen.
The triazines of formula I wherein Rxe2x80x2 is other than a lower alkyl group substituted by a C(O)R wherein Rxe2x80x3 is a hydrazine or methylhydrazine group can be prepared by the synthetic route shown below in Scheme I.
In the synthetic route shown as Scheme I, the appropriate pyruvic acid derivative of formula II wherein Rxe2x80x2* is a cycloalkyl group; an aralkyl group wherein the alkyl portion contains from 1 to 6 carbon atoms, and the aryl portion is optionally substituted by 1 to three hydroxy, lower alkoxy or nitro groups; a heteroaryl group containing from 1 to 3 heteroatoms selected from the group consisting of sulfur, oxygen and nitrogen; a group of the formula 
xe2x80x83wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group;
a lower alkyl group, optionally substituted by hydroxy or a group of the formula 
wherein Rxe2x80x3 is hydroxy, a lower alkoxy group, an aryl group optionally substituted by a lower alkoxy group, and X is a hydroxy, ethoxy or sodium salt of the hydroxide, is reacted with a 1,3-diaminoguanidine acid addition salt to afford the desired compound of formula I wherein Rxe2x80x2 is other than a lower alkyl group substituted by a C(O)Rxe2x80x3 wherein Rxe2x80x3 is a hydrazine or methylhydrazine group. This reaction is typically conducted in an alkanol or aqueous alkanol solvent, at the reflux temperature of the solvent system. Typical reaction times vary according to the precise nature of the particular reactants, but are usually in the range of 12-36 hours.

The compounds of formula I wherein Rxe2x80x2 is a lower alkyl group substituted by a C(O)Rxe2x80x3 wherein Rxe2x80x3 is a hydrazine or methylhydrazine group can be prepared by the synthetic route shown below in Scheme II.
In this synthetic route, a compound of formula I wherein Rxe2x80x2 is an alkoxycarbonyl group is utilized as the starting material to prepare the corresponding compound of formula I wherein Rxe2x80x2 is a lower alkyl group substituted by a C(O)Rxe2x80x3 wherein Rxe2x80x3 is a hydrazine or methylhydrazine group. Typically, the compound of formula I wherein Rxe2x80x2 is an alkoxycarbonyl group is treated with anhydrous hydrazine or methylhydrazine and water at reflux temperatures. Typical times vary from about 0.5 to 3 hours. Preferably, the resultant product, which is the free base, is converted to the acid addition salt, preferably one such as the hydrochloride, without isolation. The salt form is then crystallized to give the desired product.

The compounds of formula I wherein Rxe2x80x2 is a lower alkyl group substituted by a hydroxy group can also be prepared by the synthetic route shown in Scheme III. In this scheme, a dihydro4,4-dimethyl-2,3-furandione is utilized in place of the compound of formula II of Scheme I. Other reaction conditions are generally similar. Thus afforded are the particular hydroxyalkyl compounds wherein the lower alkyl group is an ethyl or propyl group, optionally substituted by one or more lower alkyl groups.
